The nucleus is the inner sanctuary of the eukaryotic cell, keeping the genetic information enclosed by the nuclear envelope (NE). The NE consists of two concentric membrane bilayers fused at specific sites to form circular openings, each occupied by a nuclear pore complex (NPC). As the only gateway into and out of the nucleus, NPCs play a critical role in cellular homeostasis, but they are more than just transport complexes. We are beginning to appreciate that the NPC also serves as a reference point to organize the nucleus and plays a pivotal role in gene regulation. This research program focuses on determining the molecular architecture of the enormous ~40-80 MDa NPC. Attaining this goal will provide a structural basis to interrogate its myriad functions in transport, gene regulation, and nuclear organization. Importantly, aberrant function of the NPC and its constituents is a primary cause of many human diseases, including leukemia, other cancers, autoimmune diseases, cardiomyopathies, and a variety of viral infections. The NPC is a modular structure, composed of ~30 different proteins, known as nucleoporins that arrange in multiple copies around a central eightfold rotational axis. Subcomplexes of 2-10 nucleoporins hierarchically build up the NPC. Two of these subcomplexes, the heteromeric Y- and Nic96-complexes, organize the majority of architectural nucleoporins and establish the stable scaffold of the NPC. Crystallographic analysis of these subcomplexes by different labs combined with cryo-electron tomographic reconstitution has recently led to a first, vague assembly model. Here, we propose to fully structurally characterize the scaffold components of the NPC. Complemented by biochemical and cell biological methods, we hypothesize that we will generate the data to build a detailed structure of the NPC, the largest assembly structure in the eukaryotic cell. The proposal is centered on establishing the major building blocks of the NPC at near-atomic resolution, using a combination of X-ray crystallographic and electronmicroscopic techniques. A second focus of this study is to establish the interactome of all nucleoporins using experimental methods. This information will be critical in order to build a robust model of the NPC. Such a 3D rendition of the NPC can then be used to interrogate the NPC functions at a detailed, molecular level.